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Rapid-Quench and Isotope-Trapping Studies on Fructose- 1,6-bisphosphataset Jubrail F. Rahil, Margaret M. de Maine,t and Stephen J. Benkovic*
ABSTRACT: Rapid-quench kinetic measurements yielded presteady-state rate data for rabbit liver fructose- 1,6-bisphosphatase (FBPase) (a tetramer of four identical subunits) that are triphasic: the rapid release of Pi (complete within 5 ms), followed by a second reaction phase liberating additional Pi that completes the initial turnover of two or four subunits of the enzyme (requiring 100-150 ms), and a steady-state rate whose magnitude depends on the [a-Fru-1,6-P2]/ [FBPase] ratio. With Mg2+ in the presence of excess a-fructose 1,6bisphosphate (a-Fru- l,6-P2) all four subunits turn over in the pre steady state; with Mn2+ only two of the four are active. Thus the expression of half-site reactivity is a consequence of the nature of the metal ion and not a subunit asymmetry. In the presence of limiting a-anomer concentrations only two of
the four subunits now remain active with Mg2+as well as with Mn2+in the pre steady state. However, so that the amount of Pi released can be accounted for, a /3 a anomerization or direct /3 utilization is required at the active site of one subunit. Such behavior is consistent with the two-state conformational hysteresis displayed by the enzyme and altered affinities manifested within these states for a and /3 substrate analogues. Under these limiting conditions the subsequent steady-state rate is limited by the /3 a solution anomerization. These data in combination with pulse-chase experiments permit evaluation of the internal equilibrium, which in the case of Mg2+is unequivocally higher in favor of product complexes and represents a departure from balanced internal substrate-product complexes.
Fructose-1,6-bisphosphatase(EC 3.1.3.1 1, D-fructose-1,6bisphosphate 1-phosphohydrolase, FBPase') (Benkovic & de Maine, 1982) catalyzes the hydrolysis of D-fructose 1,6-bisphosphate (Fru-l,6-P2) to D-fructose 6-phosphate (Fru-6-P) and inorganic phosphate (Pi). It is a tetrameric protein with four identical subunits (Traniello et al., 1971) and requires Mg2+,Mn2+, Zn2+,or Co2+for its catalytic activity. Kinetic studies with Mn2+ on the rabbit liver enzyme demonstrated its high stereospecificity for the a anomer of Fru-1,6-P2 (Frey et al., 1977). However, the stereospecificity appears anomalous in that the enzyme binds both the CY and /3 anomers of methyl D-fructofuranoside 1,6-bisphosphate (Ki = 7.2 pM and 1.7 pM, respectively) (de Maine & Benkovic, 1972). An explanation is provided by the existence of hysteretic active and resting forms of FBPase (de Maine & Benkovic, 1979). Results from pre-steady-state kinetic investigations employing a phenol red
indicator method on FBPase with Mg2+ fitted the proposed hysteresis. They suggested a reaction sequence in which binding of the substrate is followed by a first-order conformational change prior to the establishment of the steady state. The conformational change (manifest by proton release) is followed by a rapid proton uptake of a magnitude corresponding to the release of Pi from a molecule of Fru- 1,6-P2 by each subunit of the FBPase. After breakdown of the remaining substrate at the steady-state rate the catalytic cycle ends with the decay of the enzyme to its initial unreactive state (Benkovic et al., 1979). The present study concerns itself with molecular events that occur in the transient phase, namely, (1) verification that Pi is indeed released in this phase, (2) the stoichiometry of Pi release relative to conditions where Fru- 1,6-P2as the a anomer is less than FBPase subunit concentration, (3) the influence
From the Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802. Received December 1 , 1981. This investigation was supported by a grant from the National Institutes of Health, US.Public Health Service (GM 13306). 'Awardee of an Eloise Gerry fellowship from Sigma Delta Epsilon.
I Abbreviations: FBPase, fructose-l,6-bisphosphatase; Fru-1 ,6-P2, fructose 1,6-bisphosphate; Fru-6-P, fructose 6-phosphate; Pi, inorganic phosphate; EDTA, ethylenediaminetetraacetic acid; NADP', nicotinamide adenine dinucleotide phosphate; Tris, tris(hydroxymethy1)aminomethane; NMR, nuclear magnetic resonance.
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0006-2960/82/0421-3358$01.25/0 0 1982 American Chemical Society
FRUCTOSE-BISPHOSPHATASE
of various divalent metal ions on the events in the transient phase, and (4) the internal equilibrium between Fru-1 ,6-P2 and Fru-6-P plus Pi at the FBPase active site. Experimental Procedures Materials Fru- 1,6-P2, Fru-6-P, NADP', EDTA, phosphohexose isomerase, glucose-6-phosphate dehydrogenase, and phosphofructokinase were purchased from Sigma Chemical Co. Inorganic salts were reagent grade. Tris buffers were prepared from Trizma base (Sigma, reagent grade). Liquiscint scintillation cocktail was obtained from National Diagnostics. Young fasted rabbit livers were purchased from Pel-Freez Biologicals. Carrier-free [y-32P]ATP(500 pCi), obtained from New England Nuclear, was reacted with Fru-6-P and phosphofructokinase to make [ 1-32P]Fru-1,6-P2,which was purified by chromatography on a Dowex 1-C1 column (Bartlett, 1959). Methods FBPase. Neutral rabbit liver FBPase was purified from the frozen livers of young, 24 h fasted rabbits by the procedure of Ulm et al. (1975) as modified by S. J. Benkovic et al. (1974). One sharp protein band was observed for the purified enzyme in both disc and sodium dodecyl sulfate gel electrophoresis as described previously (Benkovic, P. A., et al., 1974). Protein concentrations were determined on the basis of the standard absorbance of 0.71 for a 1.0 mg/mL solution. The enzyme was assayed spectrophotometrically by following the rate of NADPH production at 340 nm in the presence of excess phosphohexose isomerase and glucose-6phosphate dehydrogenase. The routine assay solution for the activity at 25 "C contained 50 mM Tris-HC1 (pH 7.6), 5 mM MgC12, 0.1 mM EDTA, 0.1 mM NADP+, 7.6 units of phosphohexose isomerase, 1.7 units of glucose-6-phosphate dehydrogenase, and 0.1 mM Fru-1 ,6-P2 in a total volume of 1 mL. When Mn2+ was utilized as the cofactor, 0.5 mM MnCl, was used with no EDTA.2 This assay provided a rate that yielded the steady-state rate when adjusted for the higher enzyme concentration in the rapid-quench systems. Extraction Analysis of [32P] Pi. Inorganic phosphate was separated from the sugar phosphates by adapting the Jencks & Gilchrist (1964) modification of the Martin & Doty (1949) procedure. A known volume of the quenched reaction solution (0.2 mL) was added to 0.3 mL of water-saturated isobutyl alcohol. The mixture was vortexed for 30 s. To this mixture was added 0.3 mL of a solution made from 2 g of ammonium molybdate, 5.55 mL of concentrated H2S04, and water to make 100 mL. After a vigorous vortexing for 60 s the two layers were separated by low-speed centrifugation. Equal volumes (1 50 pL) of each layer were added to 3 mL of Liquiscint scintillation cocktail. 32Pradioactivity was measured with a Beckman LS 8100 liquid scintillation spectrometer. Comparison of the radioactivity in the organic phase to that of the aqueous gave the relative distribution of 32Pas Pi and as Fru-1 ,6-P2. Measured values of radioactivity were adjusted for the actual volumes of the two layers. The counts of the organic layer (containing the [32P]Pi) were further corrected for trace Pi in the Fru- 1,6-P2and for extraction efficiency. The The pronounced activation of the Mg2+assay by EDTA has been attributed to the removal of inhibitory Zn2+through chelation (Nimmo & Tipton, 1975). In contrast EDTA at 0.5 mM MnZ+has a negligible effect (
